The reinforcing properties of addictive drugs result from their effects on the mesolimbic dopamine system, which projects from the ventral tegmental area to the nucleus accumbens. Dopamine plays a central role in modulating glutamatergic inputs to medium spiny neurons, the major output neuron of the nucleus accumbens. It was recently shown that dopamine neurons release glutamate in the nucleus accumbens, in addition to their canonical release of dopamine. Since glutamate transmission in the nucleus accumbens is altered in response to chronic exposure to drugs of abuse, it is critical to understand how the release of glutamate from dopamine terminals is regulated. Previous work examining corelease used electrophysiology to measure glutamate- mediated postsynaptic currents and electrochemistry to measure the extracellular concentration of dopamine. However, it is unclear whether the extracellular dopamine measured with electrochemistry is the same pool of dopamine that drives activation of synaptic dopamine receptors. We recently described a new approach to measure synaptic dopamine transmission using overexpression of a G-protein activated inward rectifier potassium (GIRK) channel. Since endogenous D2-receptors efficiently couple to these GIRK channels, the outward potassium current can be used as a sensor of D2-receptor activation. Thus for the first time, it will be possible to measure dopamine- and glutamate-mediated postsynaptic currents resulting from simultaneous release of the two transmitters from dopamine terminals. The goal of this proposal is to determine the functional organization of dopamine and glutamate cotransmission in the nucleus accumbens. Aim 1 will examine whether dopamine and glutamate can be stored in separate pools of vesicles by determining whether dopamine- and glutamate-mediated postsynaptic currents are differentially regulated. Aim 2 will assess whether dopamine and glutamate are capable of being stored in the same synaptic vesicles by pharmacologically and genetically manipulating the activity of vesicular neurotransmitter transporters. Technical training in this proposal will inclue learning to record and analyze rapid excitatory postsynaptic currents, learning to perform fast scan cyclic voltammetry (FSCV) to measure the concentration of extracellular dopamine, learning to combine FSCV with electrophysiology in simultaneous recordings, and continuing to develop skills required to maintain a colony of transgenic mice. Overall, this proposal will test the hypothesis that dopamine and glutamate, while they may be predominantly segregated to different pools of vesicles, can be released from the same synaptic vesicles in dopamine terminals in the nucleus accumbens. This will add to our understanding of how dopamine neurons are capable of transmitting heterogeneous signals to postsynaptic targets.